QUANTITATIVE MICROSPECTROPHOTOMETRICAL INVESTIGATIONS ON ADAMS TRYPTOPHAN REACTION

Chemical and microspectrophotometrical investigations were carried out in order to make use of the p-dimethylaminobenzaldehyde-nitrite-reaction (Adams) for quantitative measurements of the tryptophan content in histological sections. The studies were performed using zymogen granules of the guinea pig pancreas and included comparative chemical tests with free tryptophan, tryptophan containing dipeptides and various proteins exhibiting different tryptophan contents. The blue pigment appearing after application of the Adams-reaction to the chemical substances and histological sections studied shows 2 absorption maxima, with respect to free tryptophan at wave lengths 430 and 590 nm. Protein bound tryptophan can shift the second peak to higher wave lengths. Tryptophan, glycyl-L-tryptophan, lysozyme, α-chymotrypsin and γ-globulin were found to fulfil the law of Lambert-Beer-Bouguer whereas ovalbumin and bovine serum albumin fail to show a linear relationship between the tryptophan concentrations put in and the extinction. These results lead to the conclusion that the amount of the blue pigment obtained with the p-DMAB-nitrite-reaction is dependent on the protein used, and that the extinction measured might be related to the molecular protein structure. Microspectrophotometrical evaluations of the zymogen granules confirm the validity of the law of Lambert-Beer-Bouguer with respect to the p-DMAB-nitrite-reaction, too. Varying several reactive factors (temperature, time of condensation or oxydation, HCl-concentration) the Adams' method is found to give highly constant results. Only lowering of the HCl-concentration down to 20% or more leads to a decrease of the extinction values. Contrary to it, the reaction is most sensitive to fixatives others than 4% formol or formol alcohol (2:1, v/v). Acid- and sublimat-containing fixatives suppress or decrease, respectively, the formation of the blue pigment whereas Regaud's fixative containing potassium bichromate influences the reaction to a lesser degree. All the extinction values obtained were well reproducible in repeated reaction procedures. Considering the results referred to above the p-DMAB-nitrite-reaction can be recommended for quantitative microspectrophotometrical evaluations provided tissue components with identical protein composition are compared. © 1969 Springer-Verlag.